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Abstract

Tuberculosis relapse is a barrier to shorter treatment. It is thought that lipid rich cells, phenotypically resistant to antibiotics, may play a major role. Most studies investigating relapse use sputum samples although tissue bacteria may play an important role. We developed a nondestructive, label-free technique combining wavelength modulated Raman (WMR) spectroscopy and fluorescence detection (Nile Red staining) to interrogate Mycobacterium tuberculosis cell state. This approach could differentiate single “dormant” (lipid rich, LR) and “non-dormant” (lipid poor, LP) cells with high sensitivity and specificity. We applied this to experimentally infected guinea pig lung sections and were able to distinguish both cell types showing that the LR phenotype dominates in infected tissue. Both in-vitro and ex-vivo spectra correlated well, showing for the first time that Mycobacterium tuberculosis, likely to be phenotypically resistant to antibiotics, are present in large numbers in tissue. This is an important step in understanding the pathology of relapse supporting the idea that they may be caused by M. tuberculosis cells with lipid inclusions.

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This work was supported by the PreDiCT-TB consortium [IMI Joint undertaking grant agreement number 115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu)]. M.C. and K.D. thank the UK Engineering and Physical Sciences Research Council (Grant code EP/J01771X/1) and a European Union FAMOS project (FP7 ICT, 317744) for funding. K.D. acknowledges support from a Royal Society Leverhulme Trust Senior Fellowship and the loan of a laser from M Squared Lasers. This work was supported by the Department of Health, UK. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health.